Retroviral Vector

a technology of vectors and retrovirals, applied in the field of retroviral vector production, can solve the problems of genetic instability and poor expression, the size of these constructs could negate the efficiency of cellular entry in the transient and stable vector production system, and increase the chance of the formation of replication-competent viral particles

Inactive Publication Date: 2019-07-11
OXFORD BIOMEDICA (UK) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]The present inventors have found that surprisingly, modular constructs comprising at least two of the nucleic acid components necessary for viral vector production can be effective in generating vector production cells. In particular, this has been shown to be true for the use of bacterial plasmid constructs. The modular constructs of the present invention can be used for the generation of both transient and stable vector production cells.

Problems solved by technology

Previously, it had been thought that this would be ineffective due to modular constructs, such as those of the present invention, being too large to be held by bacterial plasmids which were to be used for retroviral production, as oversized plasmids can lead to both genetic instability and poor expression.
Additionally, the size of these constructs could negate the efficiency of cellular entry in transient and stable vector production systems, and integration in stable vector production cells using typical transfection methods.
The safety and efficacy of using such plasmids for the generation of vector production cells could also be questioned as it may lead to an increased chance of the formation of replication-competent viral particles.

Method used

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Examples

Experimental program
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Effect test

example 1

n of a Full Transient Modular Plasmid

[0347]HIV-1-GFP vector was generated in HEK293T.TetR14 cells (constitutively express the TetR protein) using the full modular DNA constructs (pOXB_Transient_Modular) that is shown in FIG. 2, or by the standard transient co-transfection process using an HIV-1 GFP genome plasmid and three inducible packaging component plasmids (FIG. 1). The standard transient co-transfection process generated vector with an average GFP FACS titre of 8.7E+05 TU / ml. When the full transient modular plasmid was used, vector was generated with an average titre of 3.5E+05 TU / ml, which is only 2.5-fold lower than the standard transient co-transfection process (FIG. 3). These results suggest that the vector titres produced using the multicomponent transient transfection system or the transient modular plasmid are surprisingly similar.

[0348]PERT analysis of vector generated from harvest 2 was normalised to the known reference standard titre thereby generating a PERT predict...

example 2

n of Lentiviral Vector Production Using Modular Constructs in a Transient Transfection Process

[0350]Modular constructs shown in FIG. 5 were evaluated by the transient co-transfection system using the modular construct that was under assessment, plus remaining single component plasmids to complete the vector system. All modular constructs tested in this experiment generated vector with GFP FACS titres above 4E+04 TU / ml, confirming that all modular combinations that had been evaluated were capable of producing lentiviral vector using a transient transfection process. The process of generating GFP vector by transient co-transfection of HEK293T cells with a combination of modular constructs and single component plasmids was not optimised and was based on the optimal transient co-transfection process when four individual plasmids are utilised. Therefore, it is likely that with further optimisation the lentiviral vector yields could be improved. Parameters which may be optimised include: ...

example 3

entiviral Vector Constructs Evaluated Using the Stable Cell Line Process to Generate Pools of Packaging Cell Lines

[0351]Stable pools of packaging cell lines were generated by stable transfection using different combinations of single component plasmids or modular lentiviral vector constructs followed by a period of antibiotic selection. Antibiotic selection ensured selection of only those cells where the lentiviral vector packaging components had been integrated into the host cells genomes. Following a period of cell culture, which ensured all un-integrated plasmid DNAs had been diluted out from the cell cultures, resulting pools of packaging cells were tested for their ability to generate lentiviral vectors containing GFP following transient transfection of HIV-1 GFP genome (and Rev for PAC006) and doxycycline induction. FIG. 6 details the modular construct and single plasmid combinations that were used in the generation of each of the pools of packaging cell lines. In addition, FI...

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Abstract

A cell for producing retroviral vectors comprising nucleic acid sequences encoding: i) gag-pol; ii) env; iii) the RNA genome of the retroviral vector; and iv) optionally rev, or a functional substitute thereof, wherein at least two nucleic acid sequences are located at the same genetic locus; and wherein the at least two nucleic acid sequences are in reverse and/or alternating orientations.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority of European Patent Application No. 17210359.0, filed on Dec. 22, 2017, the entire contents of which are incorporated by reference herein.FIELD OF THE INVENTION[0002]The invention relates to the production of retroviral vectors. In particular, it relates to modular constructs comprising retroviral gene components.BACKGROUND TO THE INVENTION[0003]Gene therapy broadly involves the use of genetic material to treat disease. It includes the supplementation of cells with defective genes (e.g. those harbouring mutations) with functional copies of those genes, the inactivation of improperly functioning genes and the introduction of new therapeutic genes.[0004]Therapeutic genetic material may be incorporated into the target cells of a host using vectors to enable the transfer of nucleic acids. Such vectors can be generally divided into viral and non-viral categories.[0005]Viruses naturally introduce t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86
CPCC12N15/86C12N2740/10052C12N2740/10041C12N2740/13051C12N2740/13052C12N2740/15051C12N2740/15052C12N2740/16051C12N2740/16052
Inventor STEWART, HANNAHMAUNDER, HELENMITROPHANOUS, KYRIACOSFARLEY, DANIEL
Owner OXFORD BIOMEDICA (UK) LTD
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